The goal of this proposal is to understand how integration of signaling between MKK/ERK and Rho GTPase pathways controls melanoma progression. Preliminary studies provide novel evidence that in melanoma, Rac and Rho GTPases, along with MKK/ERK, are activated in a stage-specific manner. Active Rac promotes transformation in early but not late stages, and active RhoA promotes invasion in late, but not early stages. An important link between Rac/Rho and MKK/ERK signaling is provided by new findings that plexin B1 and sema 3C are repressed by MKK/ERK, providing novel evidence for crossregulation between MKK/ERK and plexin/semaphorin pathways. Plexin B1 has a novel tumor suppressor role in early stage cells, while suppressing migration and invasion in late stage cells. SemaSC partly shares this function, as a suppressor of migration and invasion. A second link involves RhoA-dependent induction of a protein we have named Mediator of Rho-dependent Invasion (MRDI), a novel regulator of actin stress fibers and focal adhesion events which collaborates with RhoA to promote metastatic cell invasion. Specific Aim 1 will investigate MRDI and the mechanism by which it promotes Rho-dependent cell invasion. Specific Aim 2 will examine plexin B1 and how its repression by MKK/ERK controls tumor formation and invasion in melanoma. Specific Aim 3 will investigate combinatorial interactions between MKK/ERK and Rac which may promote transformation. Specific Aim 4 will investigate sema3C-its importance for melanoma and the mechanisms involved in its repression by MKK/ERK. Our data show that melanoma is a unique model for understanding how MKK/ERK, Rac, and Rho pathways interact to promote tumor growth and invasion, and how responses to signaling mechanisms vary with cancer stages. These studies will combine innovative approaches of proteomics profiling with molecular biology, biochemistry, and cell biology to uncover novel mechanisms underlying this disease.